Micellar Stability in Biological Media Dictates Internalization in Living

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Micellar stability in biological media dictates internalization in living cells Natalia Feiner-Gracia, Marina Buzhor, Edgar Fuentes, Silvia Pujals, Roey J. Amir, and Lorenzo Albertazzi J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b08351 • Publication Date (Web): 27 Oct 2017 Downloaded from http://pubs.acs.org on October 27, 2017

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Journal of the American Chemical Society

Micellar stability in biological media dictates internalization in living cells Natalia Feiner-Gracia,†, § Marina Buzhor,∥,‡, § Edgar Fuentes,†, § Sílvia Pujals†, Roey J. Amir,*,∥,‡,# and Lorenzo Albertazzi*,† † Institute for Bioengineering of Catalonia (IBEC). The Barcelona Institute of Science and Technology, Baldiri Reixac 1521, 08028 Barcelona, Spain ∥ Department of Organic Chemistry, School of Chemistry, Faculty of Exact Sciences, Tel-Aviv University, Tel-Aviv 6997801, Israel ‡ Tel Aviv University Center for Nanoscience and Nanotechnology, Tel-Aviv University, Tel-Aviv 6997801, Israel #BLAVATNIK CENTER for Drug Discovery, Tel-Aviv University, Tel-Aviv 6997801, Israel

ABSTRACT The dynamic nature of polymeric assemblies makes their stability in biological media a crucial parameter

for their potential use as drug delivery systems in vivo. Therefore, it is essential to study and understand the behavior of self-assembled nanocarriers under conditions that will be encountered in vivo such as extreme dilutions and interactions with blood proteins and cells. Herein, using a combination of fluorescence spectroscopy and microscopy, we studied four amphiphilic PEG-dendron hybrids and their self-assembled micelles in order to determine their structurestability relations. The high molecular precision of the dendritic block enabled us to systematically tune the hydrophobicity and stability of the assembled micelles. Using micelles that change their fluorescent properties upon disassembly, we observed that serum proteins bind to and interact with the polymeric amphiphiles in both their assembled and monomeric states. These interactions strongly affected the stability and enzymatic degradation of the micelles. Finally, using spectrally-resolved confocal imaging, we determined the relations between the stability of the polymeric assemblies in biological media and their cell entry. Our results highlight the important interplay between molecular structure, micellar stability, and cell internalization pathways, pinpointing the high sensitivity of stability-activity relations to minor structural changes and the crucial role that these relations play in designing effective polymeric nanostructures for biomedical applications.

INTRODUCTION Recently, self-assembled nanomaterials such as micelles and other polymeric assemblies have been gaining increasing attention as smart drug delivery systems (DDS).1,2 The use of micelles as DDS presents several advantages: i) improved solubility of lipophilic drugs; ii) sustained release of encapsulated molecules in the body, and iii) the ability to target cells and/or tissues of interest in order to reduce side effects and increase therapeutic effectiveness. The potential and feasibility of using polymeric DDS has been recently demonstrated by the clinical approval of a micellar system: Genexol-PM.3 The system is used in Europe and Asia for the delivery of paclitaxel in patients with metastatic breast cancer and small cell lung cancer. Despite this clinical success, Genexol remains the only clinically approved micellar DDS and new strategies to enhance the performances of micellar nanocarriers are currently under investigation.4

It is clear that in addition to high micellar stability, DDS should also have a well-defined release mechanism that will allow them to selectively release their molecular cargo in a controlled manner only at the target site of disease. One very interesting possibility is to use enzymes or proteins to trigger the degradation and disassembly of micellar assemblies and release of active cargo. The overexpression of enzymes such as matrix metalloproteinases (MMPs)5 and cathepsin B6 in tumors can potentially be used to design micelles that will effectively release drugs only in the disease tissues but not in healthy ones.7 MMPs, for example, were used to release paclitaxel covalently linked to micelles formed from diblock copolymers8,9 and polymers coupled to metalloprotease sensitive peptide (PVGLIG).10 In addition, several groups, including the Wooley,11,12 Heise,13,14 Thayumanavan15–17 and Amir18–20 groups, have independently reported the utilization of esterase and ami-

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dase degradable amphiphiles to trigger micelle disassembly and cargo release. The Thayumanavan group has also explored specific protein binding for triggering the disassembly of dendritic amphiphiles.21,22 Despite this progress toward designing effective DDS, one of the crucial challenges in using self-assembled nanomaterials is their stability in the biological media, (e.g., blood and serum). When DDS are administrated intravenously, they encounter multiple biomolecular species that are present in the blood that can interact with the polymeric assemblies and affect their structure and functionality.23,24 Serum proteins are known to adsorb onto nanocarriers forming a protein corona that affects the surface properties and behavior of the polymeric assemblies.25–28 Moreover, serum proteins can prematurely disassemble the micelle and lead to nonselective release of the cargo in healthy tissues. These issues are often overlooked in the literature, most likely due to the difficulties of studying micellar assemblies in complex biological media. Previous studies on stability of micelles in fetal bovine serum (FBS), globulin, and bovine serum albumin (BSA) solutions were carried out by spectral analysis of two dyes, which form a FRET pair, encapsulated in polymeric micelle.29–32 However, this study focused only on the dye release and did not provide information on the assembly state of the micelles. Due to the dynamic nature of self-assembled delivery systems, such as micelles, another key parameter that needs to be addressed when designing DDS is the extreme dilution that these self-assembled systems face when administrated intravenously.33 Whereas many studies report the critical micelle concentration (CMC) values as an indicator of stability, these values are often measured in water or PBS and hence they do not provide information on the behavior in serum. Therefore, understanding how dilution and blood proteins affect micellar stability and degradation is of crucial importance for designing the next generation of DDS. Herein, we studied the interactions of a series of four different enzymatically degradable amphiphiles and their micellar assemblies with serum proteins to understand the stability of these systems in serum and the consequent effect on cellular internalization. The micelles were composed from PEG-dendron amphiphiles with enzymatically cleaved dendritic end-groups. Enzymatic degradation of the hydrophobic end-groups can increase the hydrophilicity of the hybrids, leading to their disassembly. The utilization of PEG-dendron hybrids as the polymeric amphiphiles granted us ultimate control over the monodispersity of the hydrophobic dendritic block,34,35 allowing fine tuning of the amphiphilicity of the hybrids to enable a high-resolution study of structure-stability relations. Furthermore, a fluorescent reporting mechanism based on excimer formation at the assembled state allowed us to distinguish between the monomer and the micelle states by obtaining fluorescent spectral information. Carrying out a

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variety of fluorescence spectroscopy measurements in protein solutions and serum allowed us to understand the stability of the different micelles in the biological media. Moreover, using spectral confocal microscopy, we were able to determine whether the labeled polymeric hybrids were internalized in their self-assembled (micellar) or disassembled (monomeric) forms, to track their intracellular localization, and to follow micelle disassembly inside the cell in real time. These data reveal the interactions of dynamic supramolecular systems with cells, paving the way for better understanding of these interactions and for rational design of micelle-based DDS with high stability, controllable release rates, and adjustable cellular internalization mechanisms. RESULTS AND DISCUSSION Design and synthesis of enzyme-responsive micelles A series of four fluorescently labeled amphiphilic PEG dendron hybrids that can report their self-assembly and disassembly through spectral response were synthesized following a previously reported methodology (Figure 1a).36 The hybrids were labeled with 7-diethylamino-3carbocy coumarin, which was shown to form excimers when the hybrids self-assemble into micelles, leading to a significant red-shift of the emission peak, from 480 nm to approximately 560 nm.36 Hence labeling the polymeric hybrids with this dye allowed us to distinguish easily between the micelles (λEm ~ 560 nm) and the nonassembled monomeric hybrids (λEm ~ 480 nm) even in complex biological media (Figure 1b, c). All hybrids were composed of dendrons with four lipophilic endgroups and differed by the type of enzymatically cleavable end-groups. Hybrids 1 – 3 had undecanoate, octanoate, or phenyl-acetate end-groups, respectively, which could be cleaved by an esterase (Figure 1c). The undecanoate end-groups of hybrid 1 were selected based on our previous results for non-labeled hybrids with similar end-groups, which showed remarkable stability toward enzymatic degradation.19 To systemically decrease the hydrophobicity of the dendrons, we synthesized hybrids 2 and 3,36 which had eight carbon atoms in each of their end-groups; the end-group of 2 was aliphatic end-groups, and that of 3 was aromatic. We expected that the dendron with the aromatic end-groups would be slightly less hydrophobic due to the higher polarity of the aromatic rings in comparison with the aliphatic chains. In addition to the esterase-responsive hybrids, we prepared hybrid 4,36 which had four phenyl acetamide end-groups that could be cleaved by the enzyme penicillin G amidase (PGA).37 All hybrids were synthesized following a recently reported modular approach36 based on accelerated divergent growth38 of the dendron. In general, the synthetic methodology was based on growing the dendron from PEG-Lysine(Boc)Fmoc after deprotection of the Fmoc group, followed by conjugation of the labeling dye at the


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last step of the synthesis. All hybrids were synthesized in high overall yields and their characterization data correlated well with the expected theoretical values (see SI).

Figure 1. (a) Molecular structure of the amphiphilic PEG-dendron hybrids and the hydrophilic hybrids formed after enzymatic degradation of the hydrophobic end-groups. PEG used was 5 kDa. (b) Schematic presentation of the monomer-micelle equilibrium and the difference in fluorescent emission wavelength due to formation of excimers only at the assembled state. (c) Fluorescence spectra at different time intervals during the enzymatic disassembly of hybrid 3 in PBS.

In addition to the four amphiphiles, hybrids 1 - 4, we also synthesized hybrid 5 with four hydroxyl endgroups,36 which is the expected degradation product of

the three esterase-responsive hybrids 1, 2, and 3 after complete enzymatic hydrolysis of all the hydrophobic end-groups. After the synthesis of the hybrids was completed, we determined their CMCs (Figure S11) by using the solvatochromic dye Nile red.39 The obtained values (in the range of 2 to 6 µM) were found to increase as the hydrophobicity of the dendrons decreased. Dynamic light scattering (DLS) and Transmission Electron Microscopy were then used to measure the size of the formed assemblies for hybrids 1 - 4, and diameters of 22 - 36 nm were observed (Figure S12 and S13), fitting well with the expected diameters of core-shell micellar assemblies. Importantly, DLS data for hydrophilic hybrid 5, showed the sizes of around 3 nm (Figure S12), which correlate well with the expected size of individual non-assembled polymeric hybrid. Interactions of monomers and micelles with serum albumin In supramolecular systems, such as micelles, monomers and assemblies are in equilibrium and are both always present during their use as DDS; therefore, it is important to study the interactions of serum proteins with both the assembled and non-assembled states. In this sense, our system is unique as the coumarin label allows identification and study of both states of the system. First, we wanted to study whether there are any interactions between serum proteins and the micelles and/or the monomeric state (Figure 2a and 2b). To do this we designed two different assays, based on either solvatochromism or Förster Resonance Energy transfer (FRET). These assays aimed to dissect the interactions of serum proteins with the monomers and the assemblies, respectively. To examine the interaction of monomers with BSA we used hybrid 4, which should have the highest hydrolytic stability of hybrids synthesized due to its amidic bonds, at a concentration slightly below its CMC. We mixed BSA (360 µM, close to blood concentration) with the hybrid and measured changes in fluorescence spectrum. At this low concentration of hybrid 4, the polymeric amphiphile is only in its monomeric form as confirmed by the emission at 480 nm in its fluorescence spectra (Figure 2c), which is characteristic for the non-assembled state.36 After BSA addition, we observed a significant increase in fluorescence emission intensity and a 15-nm blue-shift in the spectra. These alterations in the fluorescence spectra of the coumarin dye can be attributed to a solvatochromic effect due to the direct interaction between the monomer and BSA molecules, causing changes in the polarity of the microenvironment surrounding the dye upon protein binding. Our observations are in good agreement with previous reports using coumarin dyes as protein sensors.40 Similar studies using the hydrophilic hybrid 5, which can only be in the monomer form, further confirmed the interactions of the labeled hybrid with the protein (Figure S26).



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micelles were incubated with the Cy5-labeled BSA. This peak is produced by the energy transfer from the coumarin excimers inside the micelles to the Cy5 dye on the BSA, which are in close proximity. The energy transfer was further supported by the observed decrease in intensity in the fluorescence emission of the micelles only in the presence of labeled BSA as well as by the nonfluorescence emission of BSA-Cy5 alone when excited in the same conditions (Figure S27). Due to FRET’s high dependence on distance (1-10 nm), this experiment proves the direct interaction of BSA with micelles. This interaction may be produced by i) protein penetration inside the micelle due to the flexibility of the PEG shell or ii) proteins adsorption onto the PEG shell forming a protein corona.41,42 Based on both the increase in fluorescent emission of the nonassembled hybrids and the FRET results, we conclude that the BSA molecules directly and effectively interact in close proximity with both the monomeric hybrids and their micelles. We envision that these interactions may influence the stability of the system.

Figure 2. Albumin interacts with both monomer and micelles. (a) Scheme of interaction BSA with the monomer. (b) Fluorescence emission spectra of hybrid 4 at a concentration of 2 µM in the absence or presence of BSA showing a solvatochromic effect. (c) Scheme of interaction of BSA with micelle. (d) Fluorescence emission spectra of micelles of hybrid 3 (160 µM) in the presence of Cy5-labeled and unlabeled BSA show energy transfer from coumarin excimers in the micelles to the Cy5 on the BSA.

To investigate the interactions of BSA with the micellar state, we labeled BSA with the dye Cy5, which serves as a FRET acceptor for the excimer of the coumarin dyes. An energy transfer will indicate that the assembled hybrids interact with the BSA molecules, due to overlap of the fluorescence emission spectra of the coumarin excimer with the excitation spectra of the Cy5. Micelles of hybrid 3 were incubated with either native BSA or Cy5-labeled BSA (Figure 2b), and the emitted fluorescence after micelle excitation was measured. The fluorescence spectra in Figure 2d, clearly showed the appearance of a new emission peak at 675 nm when the

Supramolecular stability Supramolecular stability toward disassembly of micelles in serum-like conditions is of major importance to optimize the in vivo performance of DDS. To evaluate the potential of the studied polymeric micelles to serve as delivery vehicles for in vivo experiments in the future, we aimed to study their micellar stability by mimicking these conditions in vitro. First, we determined the stability of the four types of micelles in the presence of increasing concentrations of BSA, as albumin is the most abundant protein in the blood. The concentration of BSA was systematically increased up to 1000 µM (two fold higher than its concentration in blood), while the concentration of hybrids was kept constant at 160 µM. Fluorescence emission spectra were collected (Figure S28), and the ratios of the emission intensities for monomer to micelle were calculated (Figure 3). The obtained results showed that at low BSA concentrations ( hybrid 2 > hybrid 3 > hybrid 4. The utilization of dendrons as the hydrophobic blocks allowed us to achieve unprecedented molecular control over these structures and to fine tune their amphiphilicities. This high molecular precision enabled us to precisely evaluate structure-stability relations for these polymer-dendron hybrids. Very interestingly, we showed that the cell internalization strongly depended

on the stability of the micelles and that there are multiple possible internalization mechanisms that depend on the micellar stability. We observed that the monomeric state can intercalate with the membrane and directly enter the cell, mainly localizing at the ER. In contrast, assembled structures enter by endocytosis and are dissembled inside the endolysosomal system. Overall, hybrid 1 was the most promising system from the present study as it showed both supramolecular and covalent stability in vitro, entered the cell in its assembled state, and disassembled inside the cells to potentially release cargo. Moreover, the identification of different mechanisms of internalization for the monomer and the micelle paves the way towards the design of novel delivery strategies where the cargo could be delivered to different intracellular locations depending on the internalization mechanism. In this framework, stable micelles can be loaded non-covalently to release the payload after endocytosis, and covalent loading of monomers with drug molecules may be an effective strategy in which slightly less stable micelles disassemble outside the cell and the cell-permeability of the drug-linked monomers will result in delivery of the drug directly into the cytosol.

Scheme 1. Stability of micelle in the presence of proteins and internalization pathways elucidated in this study.

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cal imaging. This material is available free of charge via the Internet at http://pubs.acs.org.

Supporting Information. Detailed experimental information, characterization data, CMC measurements, UV and fluorescence spectra, HPLC degradation/stability data, cytotoxicity and confo-

AUTHOR INFORMATION Corresponding Author



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*Lorenzo Albertazzi, Email: [email protected] Roey J. Amir, Email: [email protected]

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Authors Contributions

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§

These authors contributed equally.

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ACKNOWLEDGMENT

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This work was financially supported by the AXA research fund (L.A.) and by the Spanish Ministry of Economy, Industry and Competitiveness (L.A., N.F. and E.F.) and by the Generalitat de Catalunya through the CERCA program. Moreover this work was supported by the Spanish Ministry of Economy, Industry and Competitiveness through the project SAF2016-75241-R (MINECO-FEDER). This research was also supported by the Israel Science Foundation (grants No. 966/14 and 2221/14). MB and RJA thank the Ministry of Science, Technology and Space od the state if Israel for their financial support.

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Micellar Stability in Biological Media Dictates Internalization in Living

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